BACKGROUND: We and others recently discovered that HIV infection can be maintained during antiretroviral therapy (ART) by proliferation of cells that were likely infected prior to initiating ART. However, it is not known how commonly such clones express HIV RNA during ART, particularly those carrying replication-competent proviruses. Such a finding could reveal that there is an important active reservoir for HIV that must be eradicated to cure the infection. The findings that expanded clones can be the source of persistent viremia(Maldarelli et al., Science 345:179-183, 2014; Simonetti et al., PNAS 113:1883-1888, 2016) indicate that at least some members of clonalpopulations can express unspliced RNA during ART and may, therefore, result in rebound viremia if ART is interrupted. We hypothesize that the majority of infected cells that persist in individuals on ART have undergone clonal expansionand are composed of members that express HIV RNA. Of note, the expression of HIV RNA is not an indication of ongoing viral replication, as the majority of these transcripts likely result from proviruses that are replication incompetent. This hypothesis leads to a model of HIV persistence during ART as consisting of a small minority of expanded clonal populations of cells that carry intact proviruses, with a small fraction of the members of these clonal populations expressing HIV RNA, leading to the production of replication-competent virus particles present in persistent viremia during ART and leading to rapid rebound when ART is interrupted. To test this hypothesis, we have begun to examine HIV expression levels in single cells in both treated and untreated individuals. It is not known what fraction of infected blood cells express HIV RNA at a given point in time, to what levels HIV is expressed in single cells, and if expression levels and profiles change with ART or remain the same. Investigating the fraction of infected cells that express HIV RNA and their levels of expression in single cells both prior to and during ART will provide a better understanding of the HIV persistence during ART and its rebound after stopping ART. To address these important questions, we are developing the single-cell HIV cell-associated RNA and DNA single-genome sequencing (CARD-SGS) method, which can be used to determine (i) the fraction of total HIV-infected cells that express HIV RNA, (ii) the levels of HIV RNA expression in single cells, (iii) the levels of HIV RNA expression in expanded clones, and (iv) the genetic relationship of these RNAs to proviral DNA. This assay uses improved isolation of intracellular RNA from total PBMCs modified from Hong et al. (J. Clin. Microbiol. 54:902-911, 2016) and endpoint dilution of HIV-expressing cells. We will use CARD-SGS to investigate changes in the HIV expression profiles over the course of treatment with ART, to identify cellular sources of persistent and rebound viremia, and to investigate the expression levels in expanded proviruses that constitute an important part of the reservoir for HIV. ACCOMPLISHMENTS: We have not only analyzed the proviral DNA but also investigated the RT-SHIV RNA extracted in bulk from tissues from infected macaques and compared these sequences to the DNA and plasma variants in the same animals. In contrast to the untreated macaques, few intracellular RNA sequences were detected in animals treated with ART for 20 weeks and most of the sequences that were detected contained frameshift mutations or large deletions, indicating that the proviruses from which they arose were not replication competent. Furthermore, these mutant RNAs did not cluster with plasma virus or the proviral DNA detected across the tissues. This finding suggests that only a minority of the infected cells that persist in blood and tissues during ART express viral RNA; furthermore, consistent with our hypothesis, the proviruses that are actively expressed are frequently defective, probably explaining why only very low levels of plasma viremia persist during ART. Most likely, the majority of productively expressing RT-SHIV-infected cells die before the 20-week ART timepoint. Although all the expressed RNA sequences detected in the treated animals were defective, no defects were found in the genomes of virus that persisted at very low levels in plasma during ART, implying that some infected cells express variants that may be replication competent and persist during ART, but at levels below the detection limit of our sampling for this study. Similarly, we have reported a replication-competent HIV-1 variant that is produced by a highly expanded cell clone and persists in the plasma of an HIV-infected human treated with ART (Simonetti et al., PNAS 113:1883-1888, 2016). Such clones are likely to be present at some level in most if not all treated patients. In addition to the expressed SHIV variants being defective, we also found that the expressed populations contained clusters of identical sequences. Populations of identical intracellular RNA sequences suggest either that multiple cells carrying identical proviruses were expressing viral RNA or that single cells (or their descendants) were undergoing higher levels of viral expression in comparison to other infected cells. The finding that the RNA variants were all defective implies that their expression is either from a proliferating cell population or from a single cell, but not from local spread through viral replication. Results of our study conducted in collaboration with Drs. Jonathan Li (Harvard University) and Brandon Keele (AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc.) suggested that expanded clones expressing HIV RNA during ART may be a source for rebound viremia after stopping ART (Kearney et al., J. Virol. 90:1369-1376, 2016). We obtained single-genome sequences from 10 ACTG study participants infected for at least 2 years who underwent analytic treatment interruption (ATI). We compared the rebounding variants to those in pre-ART plasma in all 10 participants and with on-ART PBMC-associated DNA and RNA in 7/10 participants. In 3 of these 7participants, we detected multiple, identical DNA and HIV RNA sequences during suppression on ART that exactly matched rebounding plasma HIV sequences, suggesting that clonally expanded, infected cells may be a source of rebound viremia, and hence, a reservoir for HIV. In contrast to the RT-SHIV-infected macaques, we found that the HIV RNA populations in humans during ART were diverse and were not structurally different from the proviral populations or the pretherapy virus (sequences intermingled in the phylogenetic trees). Such a difference between macaques and humans implies that higher frequencies of infected, expressing cells persist during ART in HIV-infected humans than in RT-SHIV-infected macaques. However, theN for these studies is small and the differences may actually be due to a wide range in the levels of expressing cells that could occur in both humans and macaques. Furthermore, our study in humans showed that, although some expressing cells may have resulted in rebound viremia, most did not. This finding, along with the detection of hypermutated HIV RNA sequences, implies that defective HIV proviruses are expressed during ART and may explain previous reports on the weak association between levels of HIV cellular RNA and plasma RNA (Li et al., AIDS 30:343-353, 2016), as most defective HIV proviruses would not produce virions. The results of this study contribute to our understanding of the reservoir for HIV during ART and lead to the hypothesis that the HIV reservoir includes populations of proliferating cells, some of which are already expressing HIV RNA during ART